Method development and validation for the estimation of Atorvastatin, Ezitimibe and Fenofibrate in bulk and pharmaceutical dosage forms by RP-HPLC method

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International Journal of Farmacia
Journal Home page: www.ijfjournal.com
Method development and validation for the estimation of Atorvastatin,
Ezitimibe and Fenofibrate in bulk and pharmaceutical dosage forms by RP-
HPLC method
Asfiya Abbas*1
, Varaprasad2
Jyothishmathi Institute of Pharmaceutical Science, Ramakrishna Colony, Karimnagar – 505481
*Corresponding author: Asfiya Abbas
*
Email id: asfiya_abbas@yahoo.com
ABSTRACT
A Simple, specific and sensitive an isocratic Estimation by RP-HPLC analytical Method were developed and
validated for the quantification Atorvastatin, Ezitimibe and fenofibrate in bulk and Pharmaceutical dosage forms.
Quantification was achieved by using the mobile phase (A mixture of 80 volumes of Methanol: 10 volumes of
Acetonitrile and 10 volumes of Water.). Inertsil ODS 3V column (250×4.6mm× 5µ) was used as stationary phase.
The flow rate was 1.0 ml/min. Measurements were made at a wavelength of 256nm. The average retention times for
about 2.230min for Atorvastatin, 3.840 for Ezitimibe and 5.937min for Fenofibrate.and Internal standard was found
to be 4.37& 6.70 min. The proposed method was validated for selectivity, precision, linearity and accuracy. The
assay methods were found to be linear from 50-150µg/ml. All validation parameters were within the acceptable
range. The developed methods were successfully applied to estimate the amount of Atorvastatin, Ezitimibe and
fenofibrate
Keywords: Atorvastatin, Ezitimibe Fenofibrate, RP-HPLC method, Inertsil ODS 3V column, and Methanol,
Acetonotrile, Water and Validation.
INTRODUCTION
Atorvastatin
Atorvastatin (Lipitor) is a member of the drug class
known as statins. It is used for lowering cholesterol.
Atorvastatin is a competitive inhibitor of
hydroxymethylglutaryl-coenzyme A (HMG-CoA)
reductase, the rate-determining enzyme in cholesterol
biosynthesis via the mevalonate pathway. HMG-CoA
reductase catalyzes the conversion of HMG-CoA to
mevalonate. Atorvastatin acts primarily in the liver.
Decreased hepatic cholesterol levels increases hepatic
uptake of cholesterol and reduces plasma cholesterol
levels [1].

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Structure of atorvastatin
IUPAC Name
7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-
5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-
dihydroxyheptanoate.
Description
Ezetimibe is an anti-hyperlipidemic medication which
is used to lower cholesterol levels. Specifically, it
appears to bind to a critical mediator of cholesterol
absorption, the Niemann-Pick C1-Like 1 (NPC1L1)
protein on the gastrointestinal tract epithelial cells as
well as in hepatocytes [2].
Structure of Eztimibe
IUPAC name
(3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-
fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)
azetidin-2-one
Description
An antilipemic agent who reduces both cholesterol and
triglycerides in the blood.
Structure of Fenofibrate

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IUPAC Name
Propan-2-yl 2-{4-[(4-chlorophenyl) carbonyl]
phenoxy}-2-methylpropanoate
MATERIALS AND METHODS
Instruments The chromatographic technique
performed on a Shimadzu (LC 20 AT VP) Liquid
chromatography with SPD-20A prominence UV-
visible detector and Spinchrom software, reversed
phase C18 column (Inertsil ODS 3V column
(250×4.6mm× 5µ)) as stationary phase, Electron
corporation Nicolet evolution 100, Citizen, Digital
Ultrasonic Cleaner, Shimadzu analytical balance [3],
Vacuum micro filtration unit with 0.45µ membrane
filter was used in the study. Pharmaceutically pure
sample of Atorvastatin were obtained as gift samples
from Chandra lab, Prashanthinagar, Kukatpally,
Hyderabad, India [4]. The purity of the drug was
evaluated by obtaining its melting point and ultraviolet
(UV) and infrared (IR) spectra. No impurities were
found. The drug was used without further purification.
HPLC-grade acetonitrile and Methanol from standard
reagents Pvt. Ltd.
Determination of working wavelength (Λmax)
In simultaneous estimation of two drugs isobestic
wavelength is used. Isobestic point is the wavelength
where the molar absorptivity is the same for two
substances that are interconvertible. So this
wavelength is used in simultaneous estimation to
estimate both drugs accurately [5].
RESULTS
The wavelength of maximum absorption (λmax) of
the drug, 10 μg/ml solution of the drugs in methanol
were scanned using UV-Visible spectrophotometer
within the wavelength region of 200–400 nm against
methanol as blank. The resulting spectra are shown in
the figures.
Observation
The isosbestic point was found to be 256 nm for
atorvastatin and ezitimibe and fenofibrate in
combination and was shown in figure [6].
Preparation of mobile phase
A mixture of Methanol Acetonitrile and Water in
the ratios of 80:10:10 was prepared. The mobile phase
was sonicated for 10min to remove gases.
Analysis of formulation
Preparation of samples for assay
Standard sample
Standard stock solutions of Atorvastatin, Eztimibe
and Fenofibrate (microgram/ml) were prepared by
dissolving 5 mg of Atorvastatin, 5 mg of Ezitimibe
and 80 mg of Fenofibrate dissolved in sufficient
mobile phase. After that filtered the solution using
0.45-micron syringe filter and Sonicated for 5min and
dilute to 100 ml with mobile phase (stock). Further
dilutions are prepared in 5 replicates of of 5µg/mL of
Atorvastatin, 5µg/mL of Ezitimibe and 80µg/mL of
Fenofibrate was made by adding 1 ml of stock solution
to 10 ml of mobile phase.
Tablet sample
10tablets (each tablet contains Atorvastatin-10 mg,
Fenofibrate-160 mg and Ezetimibe-10 mg) were
weighed and taken into a mortar uniformly mixed.
Test stock solutions of 5µg/mL of Atorvastatin,
5µg/mL of Ezitimibe and 80µg/mL of Fenofibrate
were prepared by dissolving weight equivalent to5 mg
of Atorvastatin, 5 mg of Ezitimibe and 80 mg of
Fenofibrate and dissolved in sufficient mobile phase
[7]. After that filtered the solution using 0.45-micron
syringe filter and Sonicated for 5 min and dilute to
100ml with mobile phase. Further dilutions are
prepared in 5 replicates of 5µg/mL of Atorvastatin,
5µg/mL of Ezitimibe and 80µg/mL of Fenofibrate was
made by adding 1 ml of stock solution to 10 ml of
mobile phase.
Calculation
The amount of theAtorvastatin, Eztimibe and
Fenofibrate present in the formulation by using the
formula given below, and results shown in above
table:
Where,
AS: Average peak area due to standard preparation
AT: Peak area due to assay preparation

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WS: Weight of Atorvastatin, Eztimibe and
Fenofibratein mg
WT: Weight of sample in assay preparation
DT: Dilution of assay preparation
METHOD VALIDATION
Linearity and range
Preparation of standard stock solution
Standard stock solutions of Atorvastatin, Ezitimibe
and Fenofibrate (microgram/ml) were prepared by
dissolving 5mg of Atorvastatin and 5mg of Ezitimibe
and 80 mg of Fenofibrate dissolved in sufficient
mobile phase. After that filtered the solution using
0.45-micron syringe filter and Sonicated for 5min and
dilute to 100 ml with mobile phase and further
dilutions were given in the table 1.
Observation
The correlation coefficient for linear curve obtained
between concentration vs. Area for standard
preparations of Atorvastatin, Ezitimibe and
Fenofibrate is 0.9985, 0.9971 and 0.9964 respectively.
The relationship between the concentration of
Atorvastatin, Ezitimibe and Fenofibrate and area of
Atorvastatin, Ezitimibe and Fenofibrate is linear in the
range examined since all points lie in a straight line
and the correlation coefficient is well within limits [8].
Precision
Method precision
Prepared sample preparations of Atorvastatin,
Ezitimibe and Fenofibrate as per test method are
injected 6 times in to the column [9].
Limit of detection
Where, σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration
curve of the analyte.
Limit of Quantification
Where,
σ = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve
of the analyte.
Accuracy
Accuracy of the method was determined by
Recovery studies. To the formulation (pre analysed
sample), the reference standards of the drugs were
added at the level of 80%, 100%, 120%. The recovery
studies were carried out three times and the percentage
recovery and percentage mean recovery were
calculated for drug is shown in table. To check the
accuracy of the method, recovery studies were carried
out by addition of standard drug solution to pre-
analysed sample solution at three different levels 80%,
100%, 120% by adding 5% of standard drug solution
in each level [10].
Specificity by direct comparison method
There is no interference of mobile phase, solvent
and placebo with the analyte peak and also the peak
purity of analyte peak which indicate that the method
is specific for the analysis of analytes in their dosage
form.
System suitability
Standard solutions were prepared as per the test
method and injected into the chromatographic system.
The system suitability parameters like theoretical
plates, resolution and asymmetric factor were
evaluated.
Robustness
Chromatographic conditions variation
To demonstrate the robustness of the method,
prepared solution as per test method injected at
different variable conditions like using different
conditions like flow rate and temperature. System
suitability parameters were compared with that of
method precision.
Ruggedness
The ruggedness of the method was studied by the
determining the analyst to analyst variation by
performing the Assay by two different analysts [11].

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RESULTS AND DISCUSSION
Discussion
A simple and selective LC method is described for the
determination of Atorvastatin, Ezitimibe and
fenofibratein tablet dosage forms. Chromatographic
separation was achieved on a reversed-phase
C18column (Inertsil ODS 3V, 5µ, 250 mm×4.6mm)
using mobile phase of a mixture of 80 volumes of
Methanol: 10 volumes of Acetonitrile and 10 volumes
of Waterwith detection of 256 nm. Linearity was
observed in the range 3-7µg /ml for Atorvastatin (r2
=0.9985), 3-7 µg /ml for Ezitimibe (r2
=0.9971) and
48-112µg /ml for Fenofibrate (r2
=0.9964) for the
amount of drugs estimated by the proposed methods
was in good agreement with the label claim.
The proposed methods were validated. The
accuracy of the methods was assessed by recovery
studies at three different levels. Recovery experiments
indicated the absence of interference from commonly
encountered pharmaceutical additives. The method
was found to be precise as indicated by the
repeatability analysis, showing %RSD less than 2. All
statistical data proves validity of the methods and can
be used for routine analysis of pharmaceutical dosage
form.
Table 1: Linearity Preparations
y = 135.09x - 72.893
R² = 0.9985
0
200
400
600
800
1000
0 2 4 6 8
Area
Conc
Linearity of Atorvastatin
y = 108.95x - 71.048
R² = 0.9971
0
100
200
300
400
500
600
700
800
0 2 4 6 8
Area
Conc
Linearity of Ezetimibe
Preparations
Volume from standard
stock transferred in ml
Volume made up in ml
(with mobile phase)
Concentration of solution(µg /ml)
Atorvastatin Ezitimibe Fenofibrate
Preparation 1 0.6 10 3 3 48
Preparation 2 0.8 10 4 4 64
Preparation 3 1.0 10 5 5 80
Preparation 4 1.2 10 6 6 96
Preparation 5 1.4 10 7 7 112

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Table 2: LOD &LOQ data for Atorvastatin, Ezetimibe and Fenofibrate
Parameter mcg (µg/ml) Area
Atorvastatin Ezetimibe Fenofibrate Atorvastatin Ezetimibe Fenofibrate
LOD 0.04 0.05 1.58 5.22 5.23 83.64
LOQ 0.12 0.15 4.78 15.82 15.83 253.85
Table 3: Recovery data for Atorvastatin, Ezetimibe and Fenofibrate
Recovery
level
Accuracy Atorvastatin Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 5 590.141 587.259 5.09 101.78
100.79%
5 594.825
5 576.811
100% 6 738.837 730.117 5.92 98.73
6 727.979
6 723.535
120% 7 867.159 868.710 7.13 101.87
7 866.597
7 872.373
Recovery
level
Accuracy Ezitimibe Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 5 444.321 455.020 5.03 100.51
100.52%
5 456.927
5 463.812
100% 6 559.362 561.839 5.96 99.34
6 572.922
6 553.232
y = 52.942x - 302.14
R² = 0.9964
0
1000
2000
3000
4000
5000
6000
0 50 100 150Area
Conc.
Linearity of Fenofibrate

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120% 7 674.588 676.231 7.12 101.71
7 671.065
7 683.040
Recovery
level
Accuracy Fenofibrate Average %
RecoveryAmount
taken(mcg/ml)
Area Average
area
Amount
recovered(mcg/ml)
%Recovery
80% 80 4035.746 4062.386 81.5517 101.94
100.58%
80 4087.727
80 4063.686
100% 96 4607.767 4643.225 94.44111 98.38
96 4641.250
96 4680.659
120% 112 5497.645 5536.840 113.6103 101.43
112 5534.686
112 5578.188
Table 4: Results of Robustness study
Parameter
Atorvastatin Ezitimibe Fenofibrate
Retention
time(min)
Tailing
factor
Retention
time(min)
Tailing
factor
Retention
time(min)
Tailing
factor
Flow Rate
0.8 ml/min
1.0 ml/min
1.2 ml/min
2.570
2.133
1.730
1.117
1.152
1.108
4.280
3.827
2.883
1.483
1.459
1.487
6.613
5.930
4.447
1.800
1.821
1.867
Wavelength
254nm
256nm
258nm
2.290
2.133
2.290
1.139
1.152
1.124
3.823
3.827
3.820
1.459
1.459
1.393
5.900
5.930
5.897
1.873
1.821
1.873
Table 5: Ruggedness data of Atorvastatin, Ezitimibe and Fenofibrate
Ruggedness Atorvastatin Ezitimibe Fenofibrate
% RSD 1.26 0.27 0.35
Assay Analyst-1 100.26 98.88 97.69
Analyst-2 98.62 98.35 98.35
Table 6: Assay Results of Atorvastin, Ezitimibe & Finofibreate
Atorvastatin
Standard Area Sample Area
Injection-1 600.684 583.390
Injection-2 598.337 593.001
Injection-3 592.210 618.630

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Injection-4 590.073 600.684
Injection-5 586.890 610.211
Average Area 593.639 601.183
Tablet average weight 240.2
Standard weight 5
Sample weight 120.1
Label amount 10
std. purity 98.6
Amount found in mg 9.99
Assay(%purity) 99.85
Ezitimibe
Standard Area Sample Area
Injection-1 481.379 508.444
Injection-2 489.799 479.379
Injection-3 471.455 497.165
Injection-4 501.290 481.379
Injection-5 478.759 499.282
Average Area 484.5364 493.1298
Tablet average weight 240.2
Standard weight 5
Sample weight 120.1
Label amount 10
std. purity 98.7
Amount found in mg 10.05
Assay(%purity) 100.45
Fenofibrate
Standard Area Sample Area
Injection-1 4058.912 4066.285
Injection-2 4059.882 4054.258
Injection-3 4069.353 4143.728
Injection-4 4071.134 4058.912
Injection-5 4054.814 4066.391
Average Area 4062.819 4077.915
Tablet average weight 240.2
Standard weight 80
Sample weight 120.1

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Label amount 160
std. purity 98.7
Amount found in mg 158.51
Assay(%purity) 99.07
Acceptance criteria
The amount of Atorvastatin, Ezitimibe and Fenofibratepresent in the taken dosage form was found to be 99.85
%, 100.45 and 99.07% respectively.
Table 7: Method precision of Atorvastatin,Ezitimibe and Fenofibrate
Atorvastatin
S.No. Rt Area
1 2.133 573.541
2 2.140 575.530
3 2.153 573.809
4 2.160 576.811
5 2.170 578.138
6 2.183 579.956
Avg 2.1565 576.298
SD 0.0186 2.507
%RSD 0.86 0.43
Ezetimibe
S.No. Rt Area
1 3.827 461.814
2 3.827 463.922
3 3.833 465.456
4 3.837 463.812
5 3.843 463.272
6 3.847 463.807
Avg 3.836 463.681
SD 0.008 1.174
% RSD 0.22 0.25
Fenofibrate
S.No. Rt Area
1 5.930 4041.508
2 5.927 4043.788
3 5.930 4044.079

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4 5.943 4063.686
5 5.953 4071.839
6 5.960 4046.164
Avg 5.941 4051.844
SD 0.014 12.683
% RSD 0.23 0.31
Table 8: Validation parameters of evaluated method
S.
No
Parameter Value Obtained of
Atorvastatin
Value Obtained of
Ezetimibe
Value Obtained of
Finofibrate
1. Accuracy (%Recovery) 100.79% 100.52% 100.58”%
2. Linearity concentrations Range
(mcg/mL)
Regression coefficient (R2
value)
3-7
0.998
3-7
0.997
48-112
0.996
3. LOD (mcg/mL) 0.04 0.05 1.58
4. LOQ (mcg/mL) 0.12 0.15 4.78
3. Precision (% RSD)
Method precision(Repeatability)
(%RSD, n = 6)
0.0-0.86 0.0-0.22
0.0-0.23
4. Robustness Met with system
suitability criteria
Met with system
suitability criteria
Met with system
suitability criteria
5. Ruggedness(%RSD analyst to
analyst variation)
1.26 0.27
0.35
LOD = Limit of detection, LOQ = Limit of quantification, %RSD = Relative standard deviation.
Fig 1: Chromatogram for standard of Atorvastatin, Ezitimibe and Fenofibrate

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CONCLUSION
A validated RP-HPLC method has been developed
for determination of Atorvastatin, Ezitimibe and
fenofibratein their bulk and combined tablet dosage
forms. The results show that the method was found to
be specific, simple, accurate, precise and sensitive.
The method was successfully applied for the
determination of both drugs in combined tablet dosage
form. In the future, this method may be applied for
routine analysis of both the drugs in API and in tablet
formulation.
Acknowledgement
The authors are highly thankful to Chandra Labs,
Kukatpally, Hyderabad, India for providing all the
facilities to carry out this work.
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